Chapter 7

antisepsis

antisepsis

use of chemical agents on living tissue (e.g., skin) to prevent the spread of microorganisms either by inhibiting their growth or destroying them.

Bactericidal or germicidal agent

an agent, physical or chemical, that kills bacteria.

Bacteriostatic agent

agent, physical or chemical, capable of inhibiting the growth of bacteria without necessarily killing them.

disinfection

the process by which most microbial forms on inanimate objects are killed without necessarily destroying saprophytes and bacterial endospores, which leads to a reduction in the number of organisms to a level that they cannot produce infection.

Sporicidal, fungicidal, viricidal

agents capable of destroying spores, fungi, and viruses, respectively.

sterilization

the process of killing or removing all microbial forms, including spores.

heating

thermal death time

is the most common physical method of sterilization.

The rate of killing is expressed in

nature of the heat

temperature and time

Several factors can affect the process of sterilization through heating.

moist heat has greater killing action than dry heat.

as temperature increases, the time taken to sterilize decreases. In other words, there is an inverse relationship between time and
temperature.

number of microorganisms

nature of microorganisms

the more microorganisms there are, the higher the temperature and the longer the duration of the process required to destroy all of them.

spore-forming microorganisms are more difficult to destroy than non-spore-forming ones.

type of material

presence of organic material

the temperature required to sterilize materials depends on the sensitivity of the material to heat. Heat-sensitive materials will require lower temperature than heat-resistant materials.

the presence of organic materials such as fats, proteins, and sugars may necessitate higher temperatures.

moist heat

types of heat

preferred over dry heat because of its more rapid killing action. Its main mechanism of action is to cause coagulation and denaturation of proteins.

pasteurization

conventional method

Temperatures below 100 °C

This is the method of destroying disease-causing organisms in milk and milk products as well as other beverages.

where the milk is heated at 60-65 °C followed by rapid cooling.

flash method

ultra-high temperature (UHT)

involves heating at 72 °C for 15 seconds followed by rapid cooling to 13 °C.

method where heating is done at 140 °C for a period of 15 seconds and 149 °C for 0.5 seconds.

vaccine bath

serum bath

inssipation

This is used to destroy contaminating bacteria in vaccine preparations. The vaccine preparation is heated in a water bath at 60 °C for 1 hour. This procedure is not sporicidal. Only the vegetative forms of the bacteria are destroyed.

This is used to inactivate bacteria contaminating serum preparations and is done by heating at 56 °C for several successive days. Similar to vaccine bath, only the vegetative forms are destroyed since higher temperatures will cause coagulation of proteins present in the serum.

This technique is used to solidify and disinfect egg-containing and serum- containing media. The culture medium is placed on the slopes of a device called inspissator and is heated at 80-85 °C for 30 minutes for 3 successive days. The basis for the method is that on the first day, vegetative forms will die and the spores that will germinate the following day will also die.

boiling

Fractional sterilization (Tyndallization)

Temperature of 100 °C

This method involves utilizing water at boiling temperature of 100 °C. It is not sporicidal and will destroy only the vegetative forms. The killing action can be enhanced by the addition of 2% sodium bicarbonate.
Certain metal articles and glasswares can be disinfected using this method for 10-20 minutes without opening the lid of the boiler.

This method is also known as intermittent sterilization and involves exposing the material to be sterilized to live steam at 100 °C for 30-90 minutes for 3 consecutive days, depending on the material to be sterilized. This sterilization method can be used to sterilize culture media such as thiosulfate-citrate-bile salts-sucrose (ICBS) and selenite broth.

Autoclave (Steam under pressure)

Temperature above 100 °C

This is the most efficient method of sterilization because it can destroy all microbial forms. The temperature for sterilizing is dependent on the pressure of the steam. When the pressure reaches 15 pounds per square inch (psi), the temperature inside the vessel reaches 121 °C. Because of the high temperature and pressure, it would take only 15-20 minutes to sterilize the material. This method is used to sterilize instruments, surgical bandages, culture media, and other contaminated materials that can withstand high temperature and high pressure.

dry heat

depends on the penetration of heat through the material to be sterilized. It is used to sterilize materials in enclosed tubes, oils, jellies, powders, and glasswares such as test tubes and Petri dishes.

red flame

open flame (flaming)

This method is used to sterilize articles like bacteriological wire loops, straight wires, tips of forceps, and searing spatulas. The materials are held over the flame of a Bunsen burner until they become red hot. It is limited only to articles that can be heated to redness in flame.

This method also makes use of the Bunsen burner or alcohol lamp. The material to be sterilized is passed over the flame several times but is not heated to redness. It is aimed at burning the organism into ashes and is used to sterilize such articles as mouths of test tubes, scalpels, glass slides, and cover slips. Only vegetative forms are destroyed. In addition, cracking of the glassware may occur.

incineration

hot air oven

infrared rays

This method is aimed at burning the organism into ashes. The contaminated material is burned using an incinerator. Articles that must be incinerated include soiled dressings and beddings, animal carcasses, and pathological material. This will result in loss of the article and hence must be used only for articles that have to be disposed. Some materials such as polystyrene emit dense smoke and must not be incinerated.

The use of the hot air oven was first introduced by Louis Pasteur.
Articles to be sterilized are placed in the oven with a temperature of 160 °C for a period of 1 hour. This can be used to sterilize metallic instruments such as forceps, scalpels, and scissors. It can also be used to sterilize certain glasswares (e.g., Petri dishes, pipettes, or flasks), and it is the only method used to sterilize powders and ointments. The disadvantage of using this method is that because air is a poor conductor of heat, then hot air will have poor penetration of the materials to be sterilized. In addition, cotton wool and paper may get slightly charred and glasses can become smoky.

In this method, the articles to be sterilized are placed in a conveyor belt and passed through a tunnel that is heated by infrared radiators. The temperature to which the materials are subjected to is 180 °C for a period of 7.5 minutes. It can be used to sterilize metallic equipment and glassware.

desiccation

freezing

filtration

This method is based on the principle of depriving the microorganism of moisture. It is used mainly for food preservation, such as in the preparation of dried fish and fruits. It may destroy vegetative forms. Endospores are resistant to drying.

is not a reliable method of sterilization because most pathogenic organisms are resistant to low temperatures. Its main use in the laboratory is for the preservation of microorganisms in a process called lyophilization or freeze-drying where the organism is rapidly frozen then dehydrated in high vacuum and stored in a vacuum-sealed container.

This is a form of mechanical sieving that does not kill microorganisms but merely separates them from the fluid. A cellulose ester filter with a pore size of 0.22-0.45 pm is used and can filter all microorganisms except viruses and the three smallest bacteria - Mycoplasma, Rickettsia, and Chlamydia. It is used for liquid solutions that will be destroyed by heat or freezing such as serum, antibiotic solutions, sugar solutions, or urea solution. This method can be used to remove bacteria from culture media or to prepare suspensions of viruses and phages.

Ultraviolet Light (UVL) Non-ionizing radiation

Ionizing radiation

radiation

the effective UVL wavelength is in the range of 200-280 nm, with 260 nm as the most effective. This corresponds with the maximum absorption of bacterial DNA. UVL acts by inducing formation of thymine-thymine dimers, resulting in lethal frameshift mutations. Microorganisms such as bacteria, viruses, and yeasts can be inactivated within seconds. It is used to disinfect hospital wards, operating rooms, laboratories, and other rooms in the hospital that need to be sterilized. The disadvantage of UV ray is that it has low penetrance. It is also limited by the lifespan of the UV bulb. In addition, there are some bacteria that have DNA repair systems that can counteract the damage done by UV rays.

ionizing rays have greater penetrance than UV rays. It exerts its effect by causing formation of free radicals that chemically interact with proteins and nucleic acids, resulting in cell death. It is not routinely used because of its potential to harm human tissues. There are two types of ionizing radiation used for sterilization purposes: electron beams and electromagnetic rays.

Electron beams

Electromagnetic rays (Gamma rays)

are particulate in nature. A linear accelerator from a heated cathode is used to generate high speed electrons. It can be used to sterilize syringes, gloves, dressing packs, food, and some pharmaceuticals. It has lower penetrance and requires sophisticated instruments.

are produced from nuclear disintegration of selected radioactive isotopes. They have greater penetrance than electron beams but require longer exposure time. The high energy radiation produced cause damage to the microorganism's nucleic acid. It is bactericidal, fungicidal, viricidal, and sporicidal. It is used commercially to sterilize disposable Petri dishes, plastic syringes, vitamins, antibiotics, hormones, fabrics and glassware.

Sonic and Ultrasonic Vibrations

Osmotic Pressure

Some bacteria can be killed after exposure to certain frequency of sound waves. Exposure to sound waves at a frequency of approximately 20,000 cycles/ second for 1 hour can kill some bacteria and viruses. High frequency sound waves act by disrupting cells. They are used to disinfect and clean instruments and to reduce microbial load.

This method is based on the principle of osmosis, so that when the concentration of the fluid surrounding the organism is altered, this will cause the bacterial cell to collapse. This is used for preservation of fruits in syrup and meats in brine.

Concentration and potency of the chemical agent.

Duration of exposure

Temperature

a higher concentration is bactericidal whereas a lower concentration may only be bacteriostatic. This is not true for alcohol. For alcohol, the effective bactericidal concentration is at 50-80%.

The longer the time of exposure to the chemical agent, the better the killing action.

A higher temperature speeds up the rate of a chemical reaction and thus accelerates killing action. However, there are also certain chemical agents that exert optimal effect at lower temperatures.

Nature of the surrounding medium

Nature of the organism

Number of organisms or size of inoculum

The pH of the medium and the presence of extraneous materials like pus or blood decreases the efficiency of the chemical agent. These materials may inactivate or lower the concentration of the chemical agent or may bind the chemical agent to its surface.

This refers to the innate resistance of the microorganism to disinfectants. Microorganisms vary in their resistance to disinfectants. Bacteria that produce endospores may be resistant to most chemical agents. Mycobacterial cell wall is lipid-rich that makes it difficult for the chemicals to penetrate it. Gram-negative bacteria have an outer membrane that confers resistance to disinfectants.

The larger the number of microorganism present, the more time needed for a disinfectant to destroy all of them.

(1) consistency (liquid or gaseous)

(2) spectrum of activity (high level, intermediate level, and low level)

(3) mechanism of action

Chemical disinfectants may be classified based on the following:

Damage to the cell membrane

can cause smaller molecules to leak out of the bacterial cell and interfere with the active transport and energy metabolism within the cell.

surface active agents

compounds have long-chain hydrocarbons that are fat-soluble and charged ions that are water-soluble. They concentrate on the surface of membranes and disrupt membrane resulting in leakage of cell components. These agents are active against vegetative microbial forms including mycobacteria as well as enveloped viruses. They are widely used as disinfectants in homes and hospitals but their activity is reduced in the presence of hard water and organic matter.

cationic agents

anionic agents

These are detergents where the fat-soluble portion is positively charged due to combination with a quaternary nitrogen atom. These are called quaternary ammonium compounds and are effective at alkaline pH.
Examples are cetrimide and benzalkonium chloride.

These are negatively charged agents that contain long chain hydrocarbons. Examples are soaps and bile salts. They remove dirt through the process of emulsification and are most effective at acidic pH.

phenolic compounds

alcohols

these act by disrupting cell membranes as well as causing precipitation of proteins and inactivation of enzymes. These are coal-tar derivatives that act as disinfectants at high concentration and as antiseptic at low concentrations. Phenols are bactericidal and fungicidal with good activity against mycobacteria but have poor activity against spores and most viruses.

disorganize the lipid structure of the cell membrane, dehydrate cells, and cause denaturation and coagulation of cellular proteins. The microbial killing property of alcohol is seen better in a 70% aqueous solution compared to absolute alcohol. The disadvantage of using alcohols is that they are skin irritants and are also flammable.

(1) acids and alkalis

(2) alcohol and acetone

(3) phenol and cresol

Denaturing agents include:

heavy metals (mercurials, silver compounds)

halogens (iodine, chlorine, hydrogen peroxide)

cause damage to the enzyme activity of bacteria. They also cause precipitation of proteins and oxidation of sulfhydryl groups. Heavy metals are mostly bacteriostatic than bactericidal.

bactericidal oxidizing agents that cause oxidation of essential sulfhydryl groups of enzymes causing inactivation of the enzymes.

formaldehyde

glutaraldehyde

Alkylating agents

formaldehyde

glutaraldehyde

is used for surface disinfection. It can be used to sterilize bedding and furniture. It is also used to kill Mycobacterium tuberculosis in sputum and fungi in athlete's foot.

is sporicidal and used as a cold sterilant in sterilizing medical equipment such as respiratory therapy machines and other equipment that can be damaged by heat. It is more potent than aldehyde.

It requires alkaline pH for its action and exposure time of at least 3 hours to be effective.

ethylene oxide

is also sporicidal and is used in the gaseous sterilization of heat-sensitive materials or equipment like heart-lung machine, respiratory and dental equipment, and polyethylene tubes in anesthesia machines. It is more potent than glutaraldehyde but slower-acting. It is highly flammable and is usually combined with 10% CO, It causes eye irritation and is mutagenic and carcinogenic.